The pituitary glycoprotein hormone, follicle stimulating hormone (FSH), is a heterodimer comprised of two non-covalently bound subunits, α and β (Pierce et al., 1981). The α-subunit is interchangeable among the hormones of this family, which include luteinizing hormone (LH), thyrotropin stimulating hormone (TSH) and chorionic gonadotropin (CG), in addition to FSH. The β-subunit, on the other hand, is unique to each hormone and is primarily responsible for the biological specificity of hormone action (see FIG. 15 respectively).
Human FSH (hFSH) contains four N-linked carbohydrate moieties, two on each of the α- and β-subunits. A schematic of the carbohydrate moieties on hLH and hFSH is shown in FIG. 13. While the functional significance of these moieties is poorly understood, they are likely to be important for proper protein folding, subunit assembly and secretion of the hormone (Suganuma et al. 1989; Feng et al., 1995). The carbohydrate moieties may also be obligatory for signal transduction, although partially deglycosylated hormones show preserved receptor binding (Calve et al., 1986; Sairam et al., 1982).
Current pharmacologic formulations of hFSH include purified urinary derivatives and, more recently, recombinant human FSH (rhFSH). rhFSH is used clinically in the treatment of infertility and in gonadotropin replacement therapy. However, the recombinant protein suffers from a short serum half-life and correspondingly diminished biopotency, necessitating frequent administration and limiting its clinical usefulness. For example, rhFSH must be administered as a daily intramuscular or subcutaneous injection, often for 8 to 12 days when used for ovulation induction (LeContonnec et al., 1994). These regimens are associated with a number of side effects, including local irritation and discomfort, which result in poor compliance and a reduction in therapeutic efficacy. One possibility for overcoming this limitation is to increase or alter the glycosylation profile of the recombinant protein in a manner that would improve its pharmacokinetic profile and in vivo bioactivity (see Baird et al., 2001).
The first reported attempt to produce an improved rhFSH by increasing the glycosylation of the protein was the synthesis of a hybrid β-FSH subunit containing the carboxyterminal peptide (CTP) sequence of hCG (Fares et al., 1992). Among the glycoprotein hormones, hCG is known to have the longest circulating half-life. This has been attributed to the presence of four O-linked glycosylation sites on the CTP of its β-subunit, corresponding to amino acids 113–145 (Matzuk et al., 1990). Thus, the rationale for constructing a hybrid β-FSH subunit linked to the CTP was that the CTP would confer an increased serum half-life on the recombinant FSH protein. This prediction was supported by the finding that the protein (β-FSH-CTP) was able to dimerize with a coexpressed α-FSH subunit to produce a functional hormone with an increased half-life. Importantly, this β-FSH-CTP demonstrated similar in vitro bioactivity and substantially increased in vivo bioactivity compared with preparations of native hFSH.
Two recently published studies lend further support for the potential clinical usefulness of long-acting FSH proteins. The first study reported the results of hormone replacement therapy in a trial with hypogonadotropic hypogonadal men (Bouloux et al., 2001). The second reported the results of hormone replacement therapy in healthy, pituitary-suppressed female subjects (Duijker et al., 2002). Both trials demonstrated that the elimination half-life of β-FSH-CTP was increased relative to rhFSH and supported the prediction that long-acting FSH proteins could reduce the frequency of hormone injections required to achieve the desired clinical outcome.
In contrast with N-linked sugars, deglycosylation of O-linked moieties does not affect signal transduction, and hCG devoid of this extension maintains its in vitro bioactivity. Schematic examples of N-linked and O-linked carbohydrates are shown in FIG. 14.
Instead, the importance of the O-linked sugars lies in providing enhanced stability of the hormone in vivo. This was initially deduced from comparisons between hCG and hLH, whose biological activity and β subunits are remarkably similar but whose serum half lives are dramatically different. The β subunits of hCG and hLH share greater than 85% sequence identity through the N-terminal 113 amino acids (Pierce et al., 1981). In addition, these two hormones share a common receptor and elicit similar biologic activity following receptor binding. However, the serum half-life of hCG is almost five-times that of hLH (Porchet et al., 1995; Saal et al., 1991; Yen et al., 1968). The primary structural difference between β-hCG and β-hLH is the additional carboxy-terminal amino acids comprising the CTP sequence of β-hCG. This carboxy-terminal peptide, specifically its O-linked glycosylation sequences, is thus likely to be responsible for both the decreased metabolism and excretion of hCG, and thus also for its notably increased serum half-life over the relatively transient hLH.
The importance of the CTP in promoting hormone stability was demonstrated by the synthetic β-FSH-CTP protein discussed above. Thus, merely adding the CTP sequence to β-hFSH was sufficient to increase the biological activity of the hormone, most likely through an increase in serum-half life. Indeed, recent pharmacokinetic parameter estimates in humans have demonstrated that the β-hFSH-CTP protein has an elimination half-life of 2 to 3 times longer than that of native recombinant hFSH (Bouloux et al., 2001). Unfortunately, an early attempt to further increase the half-life and bioactivity of the β-hFSH-CTP protein by adding two tandem repeats of CTPs was unsuccessful (LaPolt et al., 1992). Thus, efforts to further improve the pharmacokinetics and bioactivity of β-FSH-CTP by adding additional CTP moieties were abandoned.